pattern recognition techniques to count and identify cultures of methanogens
(Methanospirillum hungatei and Methanosardna mazei). In anaerobic wastewater
digesters, floes and granules are bacterial aggregates whose size (Dudley et al., 1993) and
roughness (Bellouti et al., 1997) can be monitored by IA and related to the efficiency of
the reactors. Settleability of activated sludge can be characterised by the fractal
dimension of the floes (Grijspeerdt and Verstraete, 1997).
The main routine applications are dealing with viability assessment using such vital
stains as Orange Acridine, and epifluorescence microscopy (Singh, Pyle and McFeters,
1989).
The emergence of new fluorescent phylogenetic probes combined with microscopic
techniques allows the assessment of the identities and activities of bacterial cells within
complex microbial communities. Automated Confocal Laser Scanning Microscopy
(CLSM) combined with image processing techniques allowed the assessment of 3D
biofilm structures (see chapter “Biofilm Reactors” in this book) under in situ conditions
(Kuehn et al., 1998) and off line sampling from a rotating annular bioreactor (Lawrence,
Nie and Swerhone, 1998).
When Fluorescence In Situ Hybridisation (FISH) was combined with CLSM this
approach allowed visualising spatial organisations of microbes in methanogens granules
(Sekiguchi et al., 1999) of upflow anaerobic sludge blanket reactors. Properties of a
synthetic biofilm (Escherichia coli) were examined by using CLSM in combination with
fluorescent probes (Swope and Flicklinger, 1996). Combination of FISH and
microautoradiography enabled the assessment of the structure and function of bacterial
communities (Lee et al., 1999), specially in activated sludge processes (Nielsen et al.,
1999; Kawaharasaki et al., 1998).
YeastsThe yeast Saccharomyces cerevisiae is widely used in biopharmaceutical and food
processes. Some authors have developed IA techniques for sizing and counting of S.
cerevisiae (Vicente, Meinders and Teixeira, 1996; Yamashita et al., 1993; Costello and
Monk, 1985). Besides cell counting, morphological properties can be used to estimate
physiological condition of yeast cultures especially related to cell budding and division
(Hirano, 1990) and vacuolisation (Zalewski and Buchholz, 1996). A semiautomatic
method was proposed by Pons et al. (1993) to characterise the morphology of yeast cells,
(see Figure 2.3). Yeast size distributions and population kinetics (single and budding
cells, cell clusters) were determined during alcoholic fermentations.
Walsh et al. (1996) determined geometric properties of immobilised S. cerevisiae
microcolonies in alginate and carrageenan gel particles. IA provided measurements of the
cross-sectional area and aspect ratio of each microcolony and the distance of the centroid
of the microcolony from the centre of the cross-section.
New methodologies for multiphase bioreactors 2 33